The present utilization of enzymes to effect commercial processes is of considerable importance, and future utilization almost certainly will increase. One reason why enzymatic conversions are advantageous is their chemical specificity. Another advantage of enzymatic reactions is their relatively modest energy requirements. Still another advantage they possess is that their environmental impact is minimal relative to traditional chemical processes.
The desirability of commerical enzymatic processes has been an impetus for the development of immobilized enzyme systems. A homogeneous enzymatic process necessarily is performed batchwise and the enzyme usually is discarded because it is generally difficultly separable from reactants and products. This results in increased expense both because enzymes generally are an expensive material component of the process and because a batch process is usually more costly than a continuous one. In immobilized enzyme systems, the enzyme is "fixed" to solid support, thereby insolubilized. Although enzyme reactivity may be altered relative to a homogeneous enzyme process, the advantage of enzyme reuse far outweighs reduced enzyme activity. Additionally an immobilized enzyme system generally permits continuous processes for enzyme catalyzed conversions using, for example, a fixed bed. Accordingly, enzyme immobilization has been an important advance in the art of commerical enzymatic conversions.
Even with improved utilization of enzymes via immobilization it is desirable to increase productivity of the enzyme. By productivity is meant the amount of product formed per unit of enzyme. Not only is it desirable to increase total productivity, it also may be commercially desirable to increase the productivity per unit time, sometimes even at the cost of lower total productivity, because of associated lower product cost. Both total productivity and productivity per unit time often are limited by thermal denaturation of the enzyme.
Thermal denaturation of an enzyme is a phenomenon whereby the enzyme loses its activity with passage of time through a temperature induced process. Thus, at any given temperature enzyme activity may decay exponentially with time, leading to a decrease in productivity over any given time period and limiting total productivity. A commonly used index of thermal deactivation is the half-life of the enzyme; the greater the half-life, the less the thermal deactivation, or reciprocally, the greater the stability.
Productivity per unit time is influenced by the process temperature because the rate of enzymatic conversion increases with temperature, hence the productivity per unit time also increases with temperature. However, at some point thermal denaturation of the enzyme becomes an important competing process and above some optimum temperature the productivity per unit time will decrease because of thermal deactivation of the enzyme.
Thus it is readily seen that thermal deactivation of enzymes is a serious limitation on productivity in commerical enzymatic conversions. Although thermal deactivation cannot be eliminated, apparent retardation or reduction of deactivation will have important benefits, and processes employing such a method of retardation or reduction will have substantial competitive advantages.
An object of this invention is to retard or reduce the thermal deactivation of glucoamylase or amyloglucosidase (AG). On the one hand, accomplishing this objective will lead to greater productivity in the enzyme process. More specifically, accomplishing the objective according to the invention herein leads to greater reactant conversion per unit of enzyme activity by substantially increasing the usable lifetime unit of the AG. On the other hand, accomplishing the objective can lead to a greater productivity per unit time by permitting the enzymatic process to be carried out at a higher temperature.
Increasing the total productivity and productivity per unit time are substantial advantages which will inhere to an enzyme process using the invention described herein. Our invention is based on the discovery that thermal deactivation AG is retarded at high pressure. The invention based thereon is a method of enzymatically hydrolyzing certain carbohydrates, particularly starch or partially hydrolyzed starch, at a pressure greater than about 500 psig.
Information related to the effect of pressure on thermal denaturation of enzymes is sparse. Eyring and Magee, J. Cell. and Comp. Physiol., 20, 169 (1942) showed that increased pressure somewhat reduced the thermal deactivation of luciferase, a two-fold increase in stability being shown at 7000 psig relative to atmospheric pressure. In contrast, only one of three acetylcholinesterase sizeozymes was stabilized at high pressure against thermal denaturation, with less than a 50% increase in stability at 2000 psig. Millar, Grafius, Wild, and Palmer, Biophysical Chemistry, 2, 189 (1974). In further contrast, alpha-amylase (taka-amylase A) was shown to be denatured at high pressure, above about 80,000 psig. Miyagawa and Suzuki, Arch. Biochem. Biophys., 105, 297 (1964).
The observations upon which the invention claimed herein is based are remarkable on several counts. First, it is unpredictable that an increase in pressure will cause stabilization of amyloglucosidase against thermal denaturation. Secondly, the magnitude of such stabilization is without precedent. In particular, we observed an increased stabilization of over 400% at only 3000 psig.